Ca2+-sensing Transgenic Mice

Ji, Guangju; Feldman, Michael R.; Deng, Ke-Yu; Greene, Kai Su; Wilson, J.; Lee, Jane C.; Johnston, Robyn C.; Rishniw, Mark; Tallini, Yvonne N.; Zhou, Jin; Wier, W. Gil; Blaustein, Mordecai P.; Xin, Hong‐Bo; Nakai, Junichi; Kotlikoff, Michael I.

doi:10.1074/jbc.m401084200

Cited by 66 publications

(26 citation statements)

References 33 publications

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“…The 7,831-bp transgene was excised with NotI, gel purified, and injected into oocytes using standard techniques. Animals were genotyped by using primers for eGFP (18). Three lines were bred to tTA-␣MHC mice to produce double transgenic mice.…”

Section: Methodsmentioning

confidence: 99%

“…Thus, difficulties in obtaining an adequate and stable concentration of indicator molecules within cells deep in complex tissues, the incompatibility of loading procedures in the in vivo setting, and the inability to selectively load specific cell lineages constitute substantial experimental constraints on the study of multicellular, processive Ca 2ϩ signaling in complex organ function. Genetically encoded sensors of Ca 2ϩ signaling (7-13) hold great promise in this regard and have been used to study signaling in lower organisms (14-17), but they have not been effectively used in mammals in vivo because of poor intrinsic signal strength, alinearity, inadequate temperature stability, or perturbing interactions between the sensing molecule and endogenous cellular proteins (18)(19)(20)(21). Recently Pologruto et al (21) illustrated this point by a careful comparison of the performance of GCaMP1 (11), Camgaroo2 (22), and Inverse Pericam (12) with the small molecule Ca 2ϩ probes X-Rhod-5F and Fluo4-FF in brain slices, demonstrating the inability of the genetic indicators to detect physiologic, lowfrequency action potential Ca 2ϩ transients (see also ref.…”

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confidence: 99%

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Imaging cellular signals in the heart in vivo : Cardiac expression of the high-signal Ca ²⁺ indicator GCaMP2

Tallini

Ohkura

Choi

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Genetically encoded sensor proteins provide unique opportunities to advance the understanding of complex cellular interactions in physiologically relevant contexts; however, previously described sensors have proved to be of limited use to report cell signaling in vivo in mammals. Here, we describe an improved Ca 2؉ sensor, GCaMP2, its inducible expression in the mouse heart, and its use to examine signaling in heart cells in vivo. The high brightness and stability of GCaMP2 enable the measurement of myocyte Ca 2؉ transients in all regions of the beating mouse heart and prolonged pacing and mapping studies in isolated, perfused hearts. Transgene expression is efficiently temporally regulated in cardiomyocyte GCaMP2 mice, allowing recording of in vivo signals 4 weeks after transgene induction. High-resolution imaging of Ca 2؉ waves in GCaMP2-expressing embryos revealed key aspects of electrical conduction in the preseptated heart. At embryonic day (e.d.) 10.5, atrial and ventricular conduction occur rapidly, consistent with the early formation of specialized conduction pathways. However, conduction is markedly slowed through the atrioventricular canal in the e.d. 10.5 heart, forming the basis for an effective atrioventricular delay before development of the AV node, as rapid ventricular activation occurs after activation of the distal AV canal tissue. Consistent with the elimination of the inner AV canal muscle layer at e.d. 13.5, atrioventricular conduction through the canal was abolished at this stage. These studies demonstrate that GCaMP2 will have broad utility in the dissection of numerous complex cellular interactions in mammals, in vivo. atrioventricular node ͉ Ca 2ϩ imaging ͉ genetic sensor ͉ heart development ͉ fluorescent Ca 2ϩ sensor T ransient, highly regulated elevations in cytosolic free Ca 2ϩ underlie numerous cellular processes that enable organ function (1-5). In the mammalian heart, for example, efficient function depends upon the coordinated release and reuptake of Ca 2ϩ ions from intracellular organelles in millions of cells, at rates between 0.5 and 15 Hz throughout life, and even subtle dysfunctions of this process can result in cardiac arrythmias and sudden death. Whereas fluorescent imaging using purpose-designed small Ca 2ϩ -binding indicator molecules has enabled important advances in the understanding of the regulatory processes underlying Ca 2ϩ signaling in single cells (6, 7), these approaches have significant limitations in the context of a complex, multicellular organ such as the beating heart. Thus, difficulties in obtaining an adequate and stable concentration of indicator molecules within cells deep in complex tissues, the incompatibility of loading procedures in the in vivo setting, and the inability to selectively load specific cell lineages constitute substantial experimental constraints on the study of multicellular, processive Ca 2ϩ signaling in complex organ function. Genetically encoded sensors of Ca 2ϩ signaling (7-13) hold great promise in this regard and have been used t...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Imaging cellular signals in the heart in vivo : Cardiac expression of the high-signal Ca ²⁺ indicator GCaMP2

Tallini

Ohkura

Choi

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

417

418

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“…These advantages can mainly be attributed to artifi cial poly-His linker (RSET) coincidentally added in the N terminal of GCaMP2, which enabled the sensor to be fully functional at 37°C (Tallini et al, 2006). Despite having been used in characterizing Ca 2+ signaling in the heart, smooth muscle, cortical brain slice of mice (Ji et al, 2004;Tallini et al, 2006;Hires et al, 2008), the low baseline fl uorescence and poor signal-tonoise ratio hinders GCaMP2 from accurately detecting the most rapid physiological Ca 2+ signals, especially in intact organs.…”

Section: Introductionmentioning

confidence: 99%

Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement

Chen

Song

et al. 2013

Protein Cell

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“…To visualize pheromone-induced activity in a large population of neurons, we generated tetO-G-CaMP2 transgenic mouse lines (22)(23)(24). When crossed to animals carrying the OMP-IRES-tTA allele (25), G-CaMP2 expression was restricted to the neurons in the olfactory system (Fig.…”

Section: Introductionmentioning

confidence: 99%

Encoding Gender and Individual Information in the Mouse Vomeronasal Organ

Kim

et al. 2008

Science

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The mammalian vomeronasal organ detects complex chemical signals that convey information about gender, strain, and the social and reproductive status of an individual. How these signals are encoded is poorly understood. We developed transgenic mice expressing the calcium indicator G-CaMP2 and analyzed population responses of vomeronasal neurons to urine from individual animals. A substantial portion of cells was activated by either male or female urine, but only a small population of cells responded exclusively to gender-specific cues shared across strains and individuals. Female cues activated more cells and were subject to more complex hormonal regulations than male cues. In contrast to gender, strain and individual information was encoded by the combinatorial activation of neurons such that urine from different individuals activated distinctive cell populations.Pheromones are a group of chemicals critical for social communication in many animal species (1,2). Information on sex, strain, social rank, reproductive status, and terrestrial ownership is represented in the complex pheromone components in urine and bodily secretions. In mice, detection of such complex chemical signals by the vomeronasal organ (VNO) and the olfactory epithelium plays an important role in triggering endocrine changes and eliciting innate territorial aggression and mating behaviors (3-5). The rodent VNO expresses more than 250 receptors that detect pheromones and transmit the signals to the brain (6-11). It is not well understood how these neurons encode information about gender and individuals. Urine contains hundreds or even thousands of substances, only a handful of which have been identified as putative pheromones (12-16). The complexity of natural pheromone signals poses a challenge to our understanding of what information is transmitted to the vomeronasal neurons (17,18).Each vomeronasal neuron expresses only one of the ∼250 estimated pheromone receptor genes (6)(7)(8)(9)19,20), and the receptor's activation elevates intracellular calcium (21). To visualize pheromone-induced activity in a large population of neurons, we generated tetO-G-CaMP2 transgenic mouse lines (22)(23)(24). When crossed to animals carrying the OMP-IRES-tTA allele (25), G-CaMP2 expression was restricted to the neurons in the olfactory system (Fig. 1, A and B, and Movie S1). Electrophysiological properties of the G-CaMP2-expressing VNO neurons, as well as their response to pheromones, were indistinguishable from those of the controls ( fig. S1). The projection patterns of the sensory neurons and the innate mating and aggressive behaviors of the G-CaMP2 mice were also indistinguishable from those of wild-type and littermate control animals (figs. S2 to S5). In VNO slices prepared from 2-to 6-month-old male or female animals, application of diluted urine elicited an increase in fluorescence in ∼30 to 40% of G-CaMP2-positive neurons, some of which showed gender-specific responses ( Fig. 1C and Movies S2 and S3). We did not observe significant differences...

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Ca2+-sensing Transgenic Mice

Cited by 66 publications

References 33 publications

Imaging cellular signals in the heart in vivo : Cardiac expression of the high-signal Ca ²⁺ indicator GCaMP2

Imaging cellular signals in the heart in vivo : Cardiac expression of the high-signal Ca ²⁺ indicator GCaMP2

Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement

Encoding Gender and Individual Information in the Mouse Vomeronasal Organ

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Ca2+-sensing Transgenic Mice

Cited by 66 publications

References 33 publications

Imaging cellular signals in the heart in vivo : Cardiac expression of the high-signal Ca 2+ indicator GCaMP2

Imaging cellular signals in the heart in vivo : Cardiac expression of the high-signal Ca 2+ indicator GCaMP2

Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement

Encoding Gender and Individual Information in the Mouse Vomeronasal Organ

Contact Info

Product

Resources

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Imaging cellular signals in the heart in vivo : Cardiac expression of the high-signal Ca ²⁺ indicator GCaMP2

Imaging cellular signals in the heart in vivo : Cardiac expression of the high-signal Ca ²⁺ indicator GCaMP2